Receptor binding properties and C34 susceptibility of revertants.

<p><b><i>A</i></b>, CD4 binding by WT and mutated gp120 molecules. Soluble CD4 binding curves were obtained by incubating a constant amount of biosynthetically labelled WT and mutated gp120 with the indicated amounts of sCD4. gp120-sCD4 complexes were coimmunoprecipitated with mAb OKT4 and protein G-Sepharose, followed by reducing SDS-PAGE and densitometry of gp120 bands. gp120-sCD4 binding is expressed as a percentage of gp120 immunoprecipitated by IgG14. The mean ± standard deviation from 2 independent experiments is shown. <b><i>B</i></b>, Inhibition of cell-cell fusion by sCD4. Assays were conducted with 0.25 µg Env expression plasmids as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat-1003218-g001" target="_blank"><b>Fig. 1</b><b><i>F</i></b></a> except that Env-293T cells were incubated with a dilution series of sCD4 for 3.5 h prior to coculture with CD4-plus-CCR5-expressing BHK21 targets for 8 h. The data are expressed as a percentage of the maximal fusion achieved by each Env and represent the means ± standard error from 4 independent experiments. WT: blue square; T138N/L494I/K601N: red triangle; ΔN¹³⁹INN/K601N: green “X”. <b><i>C</i></b>, Inhibition of infection by sCD4-IgG2. WT, T138N/L494I/K601N and ΔN¹³⁹INN/K601N viruses were incubated with sCD4-IgG2 for 4 h at 37°C and then added to TZM-bl cells. The cells were lysed 48 h after infection and the luciferase activity present in the lysates determined. The data are expressed as a percentage of the maximal infection achieved by each virus and represent the means ± standard deviation from a single experiment performed in triplicate. Symbols are as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat-1003218-g005" target="_blank"><b>Fig. 5</b><b><i>B</i></b></a>. <b><i>D</i></b>, Utilization of CCR5 mutants in cell-cell fusion by T138N/L494I/K601N and ΔN¹³⁹INN/K601N. Assays were conducted with 0.25 µg Env expression plasmids as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003218#ppat-1003218-g001" target="_blank"><b>Fig. 1</b><b><i>F</i></b></a>. The fusion activity of each Env clone was normalized against its fusion activity with WT CCR5. Mean relative fusion activity ± standard deviation is shown from a representative experiment. CCR5 N-terminal domain: Nt, extracellular loops 1, 2, and 3: ECL1, ECL2 and ECL3, respectively. <b><i>E</i></b>, Utilization of the CCR5-Y14N coreceptor mutant in cell-cell fusion by T138N/L494I/K601N and ΔN¹³⁹INN/K601N. 293T effector cells cotransfected with 1 µg pCAG-T7 plus 0.25 µg Env expression plasmids were cocultured (16 h, 37°C) with BHK21 targets cotransfected with 1 µg pT4<i>luc</i> plus the indicated amounts of WT or Y14N-mutated pc.CCR5 prior to luciferase assay. The fusion activity of each Env clone was normalized against its fusion activity with WT CCR5. Mean relative fusion activity ± standard deviation is shown from a representative experiment. <b><i>F</i></b>, Sensitivity of T138N/L494I/K601N and ΔN¹³⁹INN/K601N to the fusion inhibitor peptide, C34. 293T effector cells cotransfected with 1 µg pCAG-T7 plus 0.25 µg Env expression plasmids were cocultured (16 h, 37°C) with BHK21 targets cotransfected with pT4<i>luc</i> plus pc.CCR5 in the presence of the indicated amounts of C34 prior to luciferase assay. The data are expressed as a percentage of cell-cell fusion activity in the absence of inhibitor (mean ± standard error; n = 3).